The plant unique ESCRT component FREE1 regulates autophagosome closure

Abstract

The energy sensor AMP-activated protein kinase (AMPK) can activate autophagy when cellular energy production becomes compromised. However, the degree to which nutrient sensing impinges on the autophagosome closure remains unknown. Here, we provide the mechanism underlying a plant unique protein FREE1, upon autophagy-induced SnRK1α1-mediated phosphorylation, functions as a linkage between ATG conjugation system and ESCRT machinery to regulate the autophagosome closure upon nutrient deprivation. Using high-resolution microscopy, 3D-electron tomography, and protease protection assay, we showed that unclosed autophagosomes accumulated in free1 mutants. Proteomic, cellular and biochemical analysis revealed the mechanistic connection between FREE1 and the ATG conjugation system/ESCRT-III complex in regulating autophagosome closure. Mass spectrometry analysis showed that the evolutionary conserved plant energy sensor SnRK1α1 phosphorylates FREE1 and recruits it to the autophagosomes to promote closure. Mutagenesis of the phosphorylation site on FREE1 caused the autophagosome closure failure. Our findings unveil how cellular energy sensing pathways regulate autophagosome closure to maintain cellular homeostasis.

Arabidopsis CaLB1 undergoes phase separation with the ESCRT protein ALIX and modulates autophagosome maturation

“…In yeast, the ESCRT-III subunit Snf7 was reported to be recruited to autophagosomal membranes by interacting with the fungi-specific protein scaffold protein Atg17 3 . In Arabidopsis , the plant-specific ESCRT component FYVE1/FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTIN 1 (FREE1) was shown to directly interact with ATG8 and ESCRT-I and was proposed to mediate the ESCRT-III recruitment to autophagosomes 7 .…”

“…Among the various ATG proteins, ATG8 populates the phagophore, coordinating the formation of autophagosomes and acting as a hub for the recruitment of receptors and adaptors which are essential for the selective recognition of autophagic cargos and help mediate the trafficking and fusion of the autophagosomes, respectively. In the final stage of autophagosome maturation, the phagophore membrane must be sealed, a process in which the endosomal sorting complex required for transport (ESCRT) machinery is implicated 3 – 7 .…”

A novel workflow for unbiased quantification of autophagosomes in 3D inArabidopsis thalianaroots

“…What is worse is that they are often only vaguely described [42]. Counting autophagosomes at individual optical 2D sections of a cell [2,3,8,44] can hardly be considered systematic. Sometimes authors use Z-stacks acquisition [6,45] which -at least in some cases -enables systematic and regular sampling of investigated cells or tissue.…”

Autophagy in a Nutshell

“…The maturation of the phagophore into a complete autophagosome follows the expansion of the vesicle into its final size and shape, and includes sealing of the open rim and clearance of associated factors. The endosomal sorting complexes required for transport (ESCRT) machinery, which promotes invagination and sealing of the intraluminal vesicle (ILV) in the multi‐vesicular body (MVB), has been reported to promote sealing [22–24], although yeast autophagosomes form even in the absence of the ESCRT‐III recycling ATPase enzyme Vps4 [25]. Whether the rim‐localized Atg2‐Atg18 complex plays a role in sealing itself beyond prior expansion is elusive, as loss of Atg2 activity leads to an upstream impairment in membrane expansion.…”